Tuberculosis (TB) remains a public health crisis and a leading cause of infection-related death globally. Although in high demand, imaging technologies that enable rapid, specific, and nongenetic labeling of live Mycobacterium tuberculosis (Mtb) remain underdeveloped. We report a dual-targeting strategy to develop a small molecular probe (CDG-DNB3) that can fluorescently label single bacilli within 1 hour. CDG-DNB3 fluoresces upon activation of the β-lactamase BlaC, a hydrolase naturally expressed in Mtb, and the fluorescent product is retained through covalent modification of the Mtb essential enzyme decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1). This dual-targeting probe not only discriminates live from dead Bacillus Calmette-Guérin (BCG) but also shows specificity for Mtb over other bacterial species including 43 nontuberculosis mycobacteria (NTM). In addition, CDG-DNB3 can image BCG phagocytosis in real time, as well as Mtb in patients’ sputum. Together with a low-cost, self-driven microfluidic chip, we have achieved rapid labeling and automated quantification of live BCG. This labeling approach should find many potential applications for research toward TB pathogenesis, treatment efficacy assessment, and diagnosis.
Tuberculosis (TB), an infectious disease caused by the slow-growing pathogen Mycobacterium tuberculosis (Mtb), kills an estimated 2 million people per year according to World Health Organization (1–3). Emergence of multidrug resistance with synergistic interaction with HIV/AIDS pandemic exacerbated this problem and reactivated a global concerted effort on TB research (4, 5). Microbial cell culture is considered as the gold standard for TB diagnosis; however, it is time-consuming—usually taking 1 to 2 months to complete because of the extremely slow growth rate of Mtb. Although nucleic acid amplification technology for detecting the DNA material from Mtb has advanced (6–8), imaging technologies that allow rapid and specific labeling of live Mtb have seen little progress (9). Since the introduction of fluorochrome staining by Hagemann in 1937, auramine O has been widely adopted for Mtb fluorescent microscopic examination. Auramine O interacts with the mycolic acids within the cell wall of acid-fast microorganisms like mycobacteria, but is not specific to Mtb and cannot discriminate viable from dead cells. Auramine O sensitivity is also undesirable and varies because of tedious staining, decolorizing, and counterstaining procedures (10, 11). Replacing this century-old technology has proven not a trivial effort: Molecular probes for live Mtb labeling have been developed to target the capsular components such as esterases (12, 13), the D-Ala-D-Ala motif of peptidoglycan (14, 15), trehalose mycolyltransesterases (16–19), and sulfatases (20), yet none has been shown to be specific for labeling Mtb. Esterases, the D-Ala-D-Ala motif, and sulfatases exist not only in mycobacteria but also in many other bacterial strains. Trehalose mycolyltransesterases are expressed in Actinobacteria phylum including mycobacteria; thus, trehalose-based probes do not have Mtb specificity either (19).
Recently, electron-deficient nitroaromatic compounds have been discovered as a new class of potent anti-TB agents (21–23) that target decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1), a periplasmic enzyme highly conserved among actinobacteria and required for the synthesis of the cell wall arabinans. Specifically, DprE1 reduces one nitro group of these compounds to a nitroso derivative and covalently modifies this nitroso to form a stable semi-mercaptal complex using the cysteine residue in the active site. This mechanism presents a possibility to design a fluorescent probe to image Mtb at the single-cell level through DprE1-mediated signal retention.
Mtb is intrinsically resistant to nearly all β-lactam antibiotics, largely because of the production of an ambler class A β-lactamase named BlaC that is highly conserved through clinical isolates (24, 25). By engineering the core structure of a β-lactam cephalosporin, we recently developed a fluorogenic probe that is specific to BlaC (26–28), but because of signal diffusion, it cannot be used for single-cell Mtb labeling. Here, we report a fluorogenic probe that targets both BlaC and DprE1 to achieve specific labeling of single live Mtb in less than 1 hour.
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